DD[E/D]-transposases catalyze the multistep reaction of cut-and-paste DNA transposition. Structurally, several DD[E/D]-transposases have been characterized, revealing a multi-domain structure with the catalytic domain possessing the RNase H-like structural motif that brings three catalytic residues (D, D, and E or D) into close proximity for the catalysis. However, the dynamic behavior of DD[E/D]-transposases during transposition remains poorly understood. Here, we analyze the rigidity and flexibility characteristics of two representative DD[E/D]-transposases Mos1 and Sleeping Beauty (SB) using the minimal distance constraint model (mDCM). We find that the catalytic domain of both transposases is globally rigid, with the notable exception of the clamp loop being flexible in the DNA-unbound form. Within this globally rigid structure, the central β-sheet of the RNase H-like motif is much less rigid in comparison to its surrounding α-helices, forming a cage-like structure. The comparison of the original SB transposase to its hyperactive version SB100X reveals the region where the change in flexibility/rigidity correlates with increased activity. This region is found to be within the RNase H-like structural motif and comprise the loop leading from beta-strand B3 to helix H1, helices H1 and H2, which are located on the same side of the central beta-sheet, and the loop between helix H3 and beta-strand B5. We further identify the RKEN214-217DAVQ mutations of the set of hyperactive mutations within the catalytic domain of SB transposase to be the driving factor that induces change in residue-pair rigidity correlations within SB transposase. Given that a signature RNase H-like structural motif is found in DD[E/D]-transposases and, more broadly, in a large superfamily of polynucleotidyl transferases, our results are relevant to these proteins as well. K E Y W O R D S distance constraint model, DNA transposon, dynamics, flexibility and rigidity, fluorescence, protein-DNA complex, transposase that includes enzymes involved in replication, recombination, DNA repair, splicing, transposition, RNA interference (RNAi), and CRISPR-Cas immunity. 15,16 The RNase H-like structural motif is schematically shown in Figure 1. It consists of a central β-sheet comprised of five β-strands (ordered 32 145 with the second β-strand B2 antiparallel to other strands) and three surrounding α-helices. Two of the three α-helices (H1 and H2) are inserted in the amino acid sequence between β-strands B3 and B4 and B4 and B5, respectively, and are located on one side of the β-sheet. The third α-helix (H3) is located on the opposite side of the β-sheet and either immediately follows β-strand B5 or is separated from it by inserted amino acid sequence of various length. The most abundant proteins in the RNHL superfamily are DD[E/D]-transposases. 15 Although there are significant differences in sequence and structure across the RNHL superfamily, the RNase H architecture represents a highly conserved core of the catalytic domain of RNHL proteins, suggesting...

compared with that of free protoheme-N-base complexes [5]. It is well known that binding of heterotropic effectors reduces the O 2 -affinity of Hb (increases in P 50 ), whereas binding of O 2 does not change the P 50 value of Hb. Effectorlinked enhancements of high-frequency thermal fluctuations of globin [6] increase the transparency of globin matrix toward migrating diatomic ligands to enhance Route [A] and to diminish Route [B], resulted in substantial (up to >10 3 -fold) decrease in the apparent O 2 -affinity (K T and K R ) or (up to 100-fold increases in P 50 ) without detectable changes in static crystallographic structures of Hb as well as the coordination/electronic structures of the hemes in T(deoxy)-and R(oxy)-Hb [5,7,8]. Allostery is an important regulatory mechanism in biology. Such mechanisms fine-tune the response of the nuclear hormone receptors (NRs) to extracellular stimuli. The structural topology of NRs consists of a mostly unstructured N-terminal (NTD)/activation function-1 domain (AF-1), a DNA binding domain (DBD) that recognizes and binds specific DNA sequences and a C-terminal ligand binding domain (LBD). Besides DNA, these domains also interact with specific factors that are subunits of the basal transcriptional machinery. Through multiple sources of data, we have observed that NRs are not simply docking sites for cellular molecules. Instead, we have characterized multiple allosteric pathways that link ligand, DNA and coactivator-binding sites. Here, we will present data from biophysical and cell-based studies on the thyroid-hormone receptor (TR). TR is a type II NR and functions as a heterodimer with the retinoid X receptor (RXR). Thyroid hormone (T3) is essential for development, differentiation, and metabolic balance in higher organisms and humans. Improper functioning of TR has been associated with pathophysiological conditions such as prostate cancer, colon cancer and hypothyroidism. Yet, the mechanism of transcriptional regulation by TR is not completely understood. Here, we present data that describes specific allosteric communication between the multiple TR domains. Specifically, we show how the AF1 influences DNA binding by the DBD and that the DNA-DBD interactions influence AF1 structural conformations. Furthermore, we show that there are direct interactions between the DBD and LBD and these are dependent on the DNA-bound state of the DBD. Our study on TR allostery also links the DNA and coactivator. Finally, propose a novel 'frustrated-fit' model to explain the negative cooperativity between TR and RXR ligands in the TR:RXR heterodimeric complex. For our studies, we utilize a combination of crystallography, experimental and theoretical biophysics and cellbased reporter gene assays. 1114-Pos Board B91 Catalyzing of Immune Peptide/MHC Class II Complex with DM MoleculeObserved by Diffracted X-ray Tracking (DXT) Major Histocompatibility complex (MHC) Class II Molecule has important role on activation of the immune system. Antigen presenting cells engulf extracellular molecules by ...

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